1
Protocadherin 12 deficiency alters the morphogenesis and the
transcriptional profile of the placenta
Christine Rampon1, Stéphanie Bouillot1, Adriana Climescu-Haulica2, Marie-Hélène
Prandini1, Francine Cand1, Yves Vandenbrouck2, Philippe Huber1,*
1
Laboratory of Vascular Pathophysiology, Inserm U882, 38054 Grenoble, France; CEA,
38054 Grenoble, France; Grenoble University, 38054 Grenoble, France;
2
Laboratory of
Informatics and Mathematics for Biology, CEA, 38054, Grenoble, France.
*Corresponding author: CEA-Grenoble, iRTSV-APV-U882, 17 rue des Martyrs, 38054
Grenoble, France. Tel 33-438 78 41 18, fax: 33-438 78 49 64, e-mail: phuber@cea.fr.
Running head: Alterations of PCDH12-deficient placentas
2
ABSTRACT
Protocadherins are transmembrane proteins exhibiting homophilic adhesive activities through
their extracellular domain. Pcdh12 is expressed in angiogenic endothelial cells, mesangial
cells of kidney glomeruli and glycogen cells of the mouse placenta. To get insights into the
role of this protein in vivo, we analyzed PCDH12-deficient mice and investigated their
placental phenotype. The mice were alive and fertile, however placental and embryonic sizes
were reduced compared to wild types. We observed defects in placental layer segregation and
a decreased vascularization of the labyrinth associated with a reduction in cell density in this
layer. To understand the molecular events responsible for the phenotypic alterations observed
in Pcdh12-/- placentas, we analyzed the expression profile of E12.5 mutant placentas in
comparison with wild types, using pangenomic chips. 2,289 genes exhibited statistically
significant changes in expressed levels due to loss of PCDH12. Functional grouping of
modified genes was obtained by GoMiner software. Gene clusters that contained most of the
differentially expressed genes were those involved in tissue morphogenesis and development,
angiogenesis, cell-matrix adhesion and migration, immune response and chromatin
remodeling. Our data show that loss of PCDH12 leads to morphological alterations of the
placenta and to notable changes in its gene expression profile. Specific genes emerging from
the microarray screen support the biological modifications observed in PCDH12-deficient
placentas.
Keywords: knockout mice, gene profiling, trophoblasts, angiogenesis
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INTRODUCTION
The placenta constitutes a physical and functional connection between the mother and the
developing embryo. It establishes an exchange system for numerous soluble compounds
between maternal and fetal bloods. Additionally, it produces hormones that promote the
maternal response to pregnancy and seems to play important roles in triggering delivery. The
placenta is itself a developing organ with successive morphological and functional
modifications adapted to the different gestation phases.
In the mouse, the placenta originates from the ectoplacental cone and the extraembryonic
ectoderm. The endothelial cells derive from the allantois (8, 16, 37). From embryonic day (E)
10, the placenta is divided in three layers associated with maternal decidual cells. The
labyrinth, located on the fetal side, is composed of an intricate array of fetal and maternal
vessels that constitute a selective barrier between the two circulation systems (1, 9). The giant
cells are located next to the uterine cells and, until E12, constitute the outermost fetal cell
layer. Between the labyrinth and the giant cells, there is a third layer, the junctional zone, also
called the “spongy layer” because of its numerous cavities. The junctional zone has been
shown to produce several hormones but its general function remains elusive. Nevertheless,
there is an absolute requirement of this layer as mutations inducing its disruption are not
compatible with embryonic survival (17, 41). The junctional zone is composed of two types
of trophoblast: the spongiotrophoblasts and the glycogen cells, recognizable by their high
glycogen contents. The glycogen cells form islets within the junctional zone that migrate from
E12.5 into the maternal decidua, beyond the giant cell line (4, 16).
Protocadherins constitute a large family of transmembrane proteins exhibiting calciumdependent homophilic adhesive properties (15, 22). As opposed to the classical cadherins,
protocadherins do not or only weakly associate with the actin cytoskeleton. Although some
4
properties of individual protocadherins have been reported, a comprehensive view of their
functions is missing.
Protocadherin 12 (PCDH12), previously called VE-cadherin 2, was initially identified in
mouse endothelial cells (43). We recently showed that its endothelial expression was more
specifically detected in angiogenic endothelium (35). In addition, PCDH12 is abundantly
expressed in placental glycogen cells and mesangial cells of renal glomeruli (35).
To gain insight into PCDH12 biological activity in vivo, we produced PCDH12-deficient
mice. The mice were alive and fertile. Furthermore, Pcdh12+/- intercrosses produced a
normal Mendelian distribution at birth (35). In this paper, we show that PCDH12-deficient
placentas and embryos are smaller than their wild type counterparts and we observed two
major morphological modifications in the mutant placentas, namely decreased vascular and
cell densities in the labyrinth and a missegregation of the labyrinthine and the junctional zone.
These general alterations of placental development prompted us to examine whether the
placental transcriptional profile was modified in absence of PCDH12. Strikingly, the
expression of 2,289 genes was significantly altered in the mutant compared to wild type
placentas. Our data show that transcriptional changes occurred in functional groups of genes
involved in tissue morphogenesis, angiogenesis, cell migration, immune response and
chromatin remodeling. Expression changes of specific genes are discussed in relation with the
biological alterations observed in PCDH12-deficient placentas.
MATERIALS AND METHODS
Animals and placenta preparation
All protocols in this study were conducted in strict accordance with the French guidelines for
the care and use of laboratory animals. Agreement 38-13 was granted by the veterinary
services of the French government to P. H. to perform the in vivo experiments described
5
herein. PCDH12-deficient mice were established on CD1 genetic background. For wild type
and Pcdh12-/- (35) placenta production, mice were mated and the day on which a vaginal plug
was found was designated 0.5. Pregnant females were killed by cervical dislocation and
conceptuses were dissected in phosphate buffer saline (PBS). Placentas and embryos were
weighed after rapid liquid draining on paper towels. Placenta volume was calculated from
measurements of diameter (D) and thickness (T) with a caliper, using the formula: π/6 x D2 x
T. Genotypes were performed on embryonic DNA as previously described (35).
Histology
For immunolocalization, tissues were snap-frozen in OCT compound and sectioned at 10-µm
with a cryomicrotome (Leica Microsystems, Wetzlar, Germany). Sections were permeabilized
in paraformaldehyde 4%, Triton 0.5%-PBS for 3 min, fixed in 4% paraformaldehyde-PBS for
20 min, saturated with 2% BSA/PBS and incubated with anti-CD31 (45) and either
peroxydase-conjugated anti-rat immunoglobulin (Biorad, Marnes-la-Coquette, France) or
alexa 488-conjugated anti-rat immunoglobulin (Invitrogen, Cergy-Pontoise, France)
antibodies, all at room temperature, using standard procedures. Peroxidase was revealed with
diaminobenzidine (DakoCytomation, Trappes, France), followed by nuclear staining with
Harris hematoxylin (Sigma-Aldrich, St-Quentin-Fallavier, France). Nuclei in fluorescent
images were stained with Hoechst 33258 (Sigma-Aldrich).
For periodic acid-Schiff (PAS) / hematoxylin (both from Sigma-Aldrich) staining, paraffin
sections were prepared using standard procedures, from paraformaldehyde prefixed tissues.
Slides were mounted in Entellan or Aquatex (VWR International, Fontenay-sous-Bois,
France) and observed with a Zeiss Axioplan microscope. Pictures were made with a digital
camera (Spot-RT, Diagnostic Instruments, Sterling Heights, Michigan) and were used to
calculate the layer surface and cell density using ImageJ software (NIH, Bethesda,
6
http://rsb.info.nih.gov/ij/) and the number of the spongiotrophoblast layer islets in the
labyrinth.
To quantify capillary length, images were processed for morphometric analysis using Image J
software. A macrocommand was edited to give the total vessel length after binarization,
skeletonization and pixel count of the CD31 staining fluorescent image.
Extraction of glycogen and measurement of glycogen content
Glycogen content of placentas was measured by the method of Lo et al. (28). Each placenta
was incubated for 30 min at 100°C with 0.5 ml of 30% KOH saturated with Na2SO4. The
solution was cooled and the glycogen was precipitated with 0.55 mL of 100% ethanol at 0°C
for 30 min. Glycogen was pelleted by centrifugation at 1,000 g for 30 min at 4°C. The pellet
was dissolved in 3 mL H2O. Aliquots were incubated for 20 min at 30°C with 0.7% phenol
and 70% H2SO4 and absorbance was measured at 490 nm. Mussel glycogen (Roche
Biochemicals, Meylan, France) was used as standard. Data were expressed as mg of glycogen
per g of wet tissue or per total placenta.
Microarray processing and analysis
Total RNAs were extracted from E12.5 placentas (5 placentas of each genotype, from 3
female and 2 male embryos, were derived from 3 litters) using Tri Reagent (Euromedex,
Souffelweyersheim, France), treated with DNase I and purified using NucleoSpin RNA Clean
up (Macherey-Nagel, Hoerdt, France). RNA concentration and integrity were tested with an
Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Massy, France).
Affymetrix chip hybridization was performed by the Institute of Genetics and Molecular and
Cellular Biology (Strasbourg, France). The amplified RNAs were labeled and hybridized to
Affymetrix mouse 430 2.0 gene chips containing over 45,000 probe sets and representing
7
more than 34,000 characterized transcripts. The arrays were scanned with a confocal laser
GeneChip scanner 3000 7G System. The resulting images were captured using GeneChip
Operating Software (all on Affymetrix instruments). The background adjustment and the data
normalization have been processed with MAS 5.0 software (Affymetrix). The scaling factors
for all arrays formed a homogeneous interval concentrated around 2.33. The exploratory data
analysis provided by the Affy package of Bioconductor (www.bioconductor.org) showed no
experimental artifacts. Boxplots, densities of log intensities, RNA digestion plots and RNA
degradation parameters are provided as supplementary data.
Full
data
sets
have
been
deposited
in
Gene
Expression
Omnibus
(GEO)
(http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO series accession number
GSE7676.
Bioinformatics analysis
Results were annotated using information provided by Affymetrix. The full data set was
reduced to 18,542 genes by discarding genes with “EST” and “unknown” annotation labels.
To generate the list of genes classified by gene ontology (GO), we used the High-Throughput
version of GoMiner (http://discover.nci.nih.gov/gominer/, (49, 50)). We then performed a
Gene Set Enrichment Analysis (GSEA) (40) for selected gene sets according to GO biological
process categories. The GSEA procedure allowed an interpretation of the gene expression
profiles, by using predefined gene sets and ranks of genes to identify significant biological
changes in microarray data sets. The family wise error rate (FWER) was used for multiple
testing corrections. It provides the probability to get a non zero false discovery rate. The heat
maps obtained from this analysis are presented as supplementary material.
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Statistical analysis
For Fig. 1-5, the statistical significance was analyzed using Student’s t test. Sample number is
indicated in figure legends. We used the “significance analysis of the microarray” (SAM)
score (44) to investigate the differentially expressed genes. The estimation of the false
discovery rate has been obtained using the q-value package available under the R language
(www.r-project.org). The computation of the q-values has been done with the default tuning
parameters as input with the exception of the pi0.method entry for which we choose the
“smoother “ method. The output value of the pi0 - the estimate of the proportion of null
hypothesis - was 0.65351.
Real-time RT-PCR
First-strand cDNAs were generated by reverse transcription from 0.5 µg of total RNA in a
total volume of 20µl with the SuperScript First strand Synthesis System (Invitrogen) using
random primers and SuperScript II Reverse transcriptase.
Real-time PCR was performed using LightCycler apparatus (Roche Diagnostics, Meylan,
France) and FastStart DNA master SYBR Green I ready to use PCR mix according to the
manufacturer’s protocol (Roche). CDNAs (25 ng) were amplified in 10 mL total volume PCR
reaction with specific primers. Gene expression was normalized using values obtained for the
housekeeping gene Gapdh as the reference gene.
Primers
used
for
PCR
amplification:
Phdal2,
5’-
gctgtttttccactccatcc
and
5’-
gtttcacggacccagagc; Gapdh, 5’- tgtgatgggtgtgaaccacgagaa and 5’- aagcatccaaccacatcaca ;
Angiopoietin 2, 5’- tacacactgacctcttccccaac and 5’- agtccacaccgccatcttctc; Tek, 5’gaacaccgaggctatttgtac and 5’- agtgtggaagctgtagtgttgg; Tie1, 5’- ggcagcttccagagtatggt and 5’tggccagcaatgttaagtca; Gja1, 5’- gagcccgaactctccttttc and 5’- ccatgtctgggcacctct ; Angiogenin2,
5’- tcagcactatgatgccaagc and 5’- tcctttgtgtgtgcaagtgg ; Decysin, 5’- aagcatccaaccacatcaca and
9
5’- tgtataggtgcacaggataggc. Primers were designed by using Integrated DNA Technologies
software (available on line at http://www.idtdna.com/Scitools/Applications/PrimerQuest) or
Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and using sequence data
from the GenBank database.
RESULTS
PCDH12 deficiency alters placenta and embryo growths
Absence of PCDH12 does not alter embryo viability, as 25.1% of homozygotes are present at
birth upon mating of heterozygous mice (35). Nonetheless, we noticed that Pcdh12-/placentas and embryos were smaller than their wild-type counterparts. This feature prompted
us to investigate several parameters of placental growth and organization. In this study,
placentas were examined at two ages: E12.5, when placenta is in a very active phase of
organogenesis and E17.5, when placenta is mature. In all following experiments, we
compared the Pcdh12-/- mice to wild types. However, it is noteworthy that heretozygotes
behaved like the wild types (not shown).
We first measured placenta weight and volume at both embryonic ages. At E17.5, but not at
E12.5, Pcdh12-/- placenta weights were indeed significantly (p < 0.001) lower than those of
wild types (Fig. 1A, B). Similarly, the volumes of E17.5 PCDH12-deficient placentas were
significantly (p < 0.001) reduced, whereas they were similar at E12.5 (Fig. 1C, D). The
volume reduction at E17.5 concerns both the diameter and the width of placentas (not shown).
The data show that Pcdh12-/- placentas did not grow significantly after E12.5, while the wild
types were still in an active phase of expansion. Furthermore, Pcdh12-/- embryo weights were
significantly lower at both ages (Fig. 1E, F). Therefore, we conclude that PCDH12 deficiency
alters both placenta and embryo growths. However, the average weight of mice of both
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genotypes aged from 2 weeks to 2 months was identical (Fig. 1G, H), thereby indicating that
Pcdh12-deficient mice recovered after birth.
We thus suspected that feto-maternal interactions were altered in absence of PCDH12.
Because embryo growth is dependent upon optimal placental activity, we focused our studies
on the placenta.
Histological analysis of Pcdh12-/- placentas
We first examined the histological organization of the different placental layers. E12.5 and
E17.5 placenta sections were prepared and labeled with the endothelial-specific anti-CD31
antibody. In the mouse placenta, the labyrinth is highly vascularized, the junctional zone is
avascular and the decidua contains large vessels; the limits of the different placental layers are
therefore clearly visualized by this technique, as illustrated in Fig. 2A. The layer surface
areas, as well as total placenta area were measured on parasagittal sections; the average
proportions are shown in Fig. 2B, C. Our results indicate that the proportions were similar
between Pcdh12-/- and wild types at both ages, thereby suggesting that growth of all three
layers was reduced in the E17.5 Pcdh12-/- placenta. The giant cell line was unmodified (not
shown).
In each layer, the cell density was evaluated after nuclear labeling and counting (Fig. 2D, E).
Interestingly, the PCDH12-deficient labyrinths contained significantly less cells per section
area than the wild types at both E12.5 and E17.5.
The labyrinth contains a very dense capillary network. As the cell density was lower in the
labyrinth, we wondered whether this network was altered in the mutant. As illustrated in Fig.
3A, B, we indeed noticed a decrease in vascular density in the mutant labyrinthine layer. We
thus measured the total capillary length in CD31 immunofluorescent images. As shown in
Fig. 3C-D, the skeletonized image obtained after processing by ImageJ software
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superimposed with the CD31-labelled vessels. Our quantitative data show that capillaries
were much less developed in the Pcdh12-/- labyrinth at both E12.5 and E17.5 (Fig. 3F, G).
This result might be in direct correlation with the lower cell density of the Pcdh12-/- labyrinth
and may also have a functional impact on embryonic development, as optimal materno-fetal
exchanges are required throughout gestation. In contrast, the vascular pattern of maternal
deciduas was similar in wild type and Pcdh12-/- placentas.
PCDH12 is an homophilic adhesive protein with high expression level in glycogen cells. We
thus wondered whether loss of PCDH12 would alter cell interactions during development.
E12.5 placentas were chosen to observe glycogen cells before their massive dissemination in
the decidua. As Pcdh12-labeling is not useable to identify the Pcdh12-/- glycogen cells,
placenta sections were labeled with PAS, which reacts with glycogen and thus highly stains
glycogen cells. The junctional zones of wild type and Pcdh12-/- mice contained a similar
pattern of glycogen cells assembled into islets with no sign of cell dissociation in the mutant
(Fig. 4A,B). Thus, PCDH12 activity is not required for glycogen cell assembly and tissue
integrity.
Projections or islets (also called “pegs”) of the junctional zone in the labyrinth may be
observed in normal placentas, even in late gestation phase (E17.5). However, we noticed that
this feature was enhanced in PCDH12-deficient placentas. Figures 4C and D show two
representative placenta sections of WT and Pcdh12-/- genotypes, respectively. The mutant
placenta exhibits numerous projections or islets within the labyrinthine zone. Enlargement of
one of this peg (Fig. 4E) shows that these structures are composed of both glycogen cells (in
dark purple) and spongiotrophoblasts (in light purple). The number of independent islets was
counted in E17.5 placentas of each genotype and the results showed a significant difference
(Fig. 4F, p < 0.001). A similar alteration was observed at E12.5 (not shown). We conclude
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that segregation of the junctional zone and the labyrinthine did not occur properly in absence
of PCDH12.
Glycogen metabolism in Pcdh12-deficient placentas
One of the major roles of the placenta is to behave as an energy supplier for the embryo.
Glycogen cells have the capacity to store huge amounts of glycogen, which can be broken
down into glucose after glucagon stimulation (6). Glycogen was extracted from whole
placentas and its concentration was measured. Glycogen concentration was significantly
higher in Pcdh12-/- placentas at E12.5 and E17.5 (Fig. 5A, B). Correspondingly, the total
amount of glycogen per placenta was increased in E12.5 Pcdh12-/- placenta (Fig. 5C);
however, glycogen amounts reached a similar level in placentas of both genotypes at E17.5
(Fig. 5D). This feature can be explained by the weight difference between wild type and
mutant placentas at this age. Thus, although the glycogen stores reached a similar level in late
gestation, glycogen production was accelerated or its accumulation was more efficient in
glycogen cells of Pcdh12-/- placentas.
Altogether, these data show that PCDH12 deficiency leads to a complex phenotype affecting
several aspects of placental development.
Gene expression profiling of PCDH12-deficient placentas
To understand the molecular events responsible for the phenotypic alterations observed in
Pcdh12-/- placentas, we analyzed the genome-wide expression profile of E12.5 mutant
placentas in comparison with wild types. RNAs from 5 placentas of each genotype were
analyzed with Affymetrix chips. The placentas derived from 3 litters. RNA quality was
examined prior to hybridization and expression data were analyzed using several
experimental and statistical criteria and gave satisfactory results (see supplemental data).
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Remarkably, from the total number of 45,101 probes, 2,289 were assigned as differentially
expressed since they present a SAM score superior to 0.75. The SAM threshold of 0.75 used
herein corresponds to the 0.95 quantile for both upregulated and downregulated genes. A
higher proportion of genes were upregulated (1,620 vs 669). Genes with unknown function
were excluded from the initial set of 45,101 probes and the bioinformatic analysis was
performed on a set of 18,542 probes.
Expression profiles obtained with DNA chips were examined by qRT-PCR for some specific
genes showing different variation levels and different signal intensities (Table 1). Fold
variations may be slightly different between the two technical approaches when the signals
were at background levels (e.g., decysin in the wild types); however, the qRT-PCR results
broadly confirm the DNA chip data.
First, a functional classification of differentially expressed genes was performed with the use
of the visualization tool GoMiner. On the basis of prior knowledge provided by the gene
ontology (GO) biological process category, we focused on classes of interest. Hence, selected
gene sets were further analyzed with the GSEA method in order to detect top differential gene
expression. In a predefined set, this method yields even small but coordinated changes (40).
As shown in Table 2, functional groups that contained most of the differentially expressed
genes were those involved in tissue morphogenesis and development, angiogenesis, cellmatrix adhesion and migration, immune response, and chromatin remodeling. All clusters
except the chromatin-remodeling group contained only up-regulated genes. The heat maps,
representing these variations in each cluster for individual samples, are shown in
Supplemental Fig. 1-5.
Independently, we examined the variations of specific genes that have previously been
involved in placenta morphogenesis or secretion, cell-cell junction and angiogenesis (Table
3). As expected, Pcdh12 expression signals reached background levels
14
For placental genes, the most significant variations were for the pleckstrin homology-like
domain, family A, member 2 (also called Ipl or Tssc3), the pregnancy-specific GP16 and 19,
the prostaglandin-endoperoxide synthase 1 (coding for cyclooxygenase 1), the solute carrier
family 21 (a prostaglandin transporter) genes. The products of these genes have pivotal roles
in placenta morphogenesis, modulation of the maternal immune system and fetal delivery (5,
7, 14, 39).
The expression of three classes of cell junction genes was altered in the mutant placenta: the
cadherin (N, P and VE), the connexin (Gja1 coding for connexin 43) and the claudin (1 and
11) families, whose products are located in adherens, gap and tight junctions, respectively.
Genes encoding for angiogenic factors and their receptors, such as the fibroblast growth
factor receptor 1, Tie 1, the transforming growth factors beta 1 and 2, and soluble Flt1, were
significantly upregulated in the mutant placentas.
Functional grouping of differentially expressed genes revealed that a large number of matrixrelated and integrin transcripts were significantly upregulated in the mutant placenta and, for
some of them, with a high Pcdh12-/- / wild type signal ratio. Table 4 shows selected genes of
this category with lowest signal value above 15 and SAM score above 0.50. Genes coding for
several collagen chains, or involved in collagen synthesis, for laminin chains, fibrillin 1 and
two fibulins are present. Most selected genes encoding matrix proteins are expressed at high
levels in the placenta. Six genes encoding integrins or proteins involved in cell-matrix
adhesion and cell migration were significantly upregulated. Cell migration also requires
matrix and integrin proteases and indeed a number of metalloproteinase family genes were
upregulated. One member, the disintegrin metalloprotease (also called decysin) gene
exhibited a 180-fold overexpression in the mutant placenta.
In addition, some miscellaneous genes, not classified by GoMiner software, were dramatically
upregulated. Table 5 shows genes with Pcdh12-/- / wild type signal ratio above 2 or below 0.5
15
with highest average value above 100 and a SAM score above 0.8. Strikingly, a gene family
for RNases or RNA-binding proteins emerged as highly differentially expressed. For
example, genes coding for eosinophil-associated ribonucleases 1 and 2 and angiogeninrelated genes were upregulated 238, 96 and 102-fold, respectively. Other unrelated genes
including a gene involved in cell proliferation, cyclin E2, exhibited a significant expression
variation.
DISCUSSION
In this paper, we show that loss of PCDH12 induced several morphological and
transcriptional changes in placental development. To our knowledge, this is the first gene
profiling study regarding a protocadherin or cadherin gene inactivation.
The embryonic growth defect was attributed to placental anomalies because the mutant mice
reached the weights of controls 2 weeks after birth. Thus, this study supports the close link
between placental and embryonic developments. As fetal growth was already retarded at
E12.5, it is likely that placental functional defects were present much earlier on.
The catch-up of PCDH12-deficient pups after birth is reminiscent of the phenotype of mice
deficient in the homeobox gene Esx1, for which placental defects, including missegregation of
labyrinthine and spongiotrophoblast layers, were observed (26). Conversely, mice with other
genetic ablations leading to placental anomalies and subsequent embryonic growth retardation
did not recover after birth the body weight of their wild type littermates (10, 47). This
observation may have important consequences in pediatrics for the management of children
born small because of abnormal placental function: a close examination of placental
phenotype and/or genetic alteration may provide indications on child thriving and may impact
medical decisions (21).
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The macroscopic morphology of PCDH12-deficient placentas was modified in two aspects:
placenta size (in late gestation) and segregation of the labyrinthine and the junctional zone.
Furthermore, the labyrinthine vasculature was much less developed; a feature that may be
related to the lower cell density in the labyrinth. As the labyrinth is subjected to a major
development between E12.5 and E17.5, and because vessel and cell densities were lower in
the mutant labyrinth, we first suspected that labyrinth growth was specifically reduced during
this time period in the mutant placenta. However, the surface proportions of the three layers
were not statistically different between the two genotypes at both ages, indicating that
placental growth retardation was general and not specific to the labyrinth. Nevertheless, it is
possible that labyrinth development influences or coordinates the growth of the junctional
zone and decidua. Alternatively, angiogenesis defects in the labyrinth and alteration of
glycogen cell behavior in the junctional zone and decidua may independently decrease the
growth rate of these layers.
The histological modifications observed herein are shared with other mouse genetic models.
For example, a missegregation of labyrinth and junctional zone was also observed in
interspecies hybrids, cloned conceptuses and after Esx1 deletion (26, 42, 48). However, this
phenotype may be caused by different molecular mechanisms (38).
Considering Pcdh12-/- histomorphological anomalies, it is striking that gene clustering
through GoMiner detected that clusters of genes involved in development, tissue
morphogenesis, adhesion and migration, as well as angiogenesis, are significantly modified
by loss of PCDH12. The majority of these genes was upregulated. Interestingly, genes of the
chromatin-remodeling cluster, mostly silencing genes, were downregulated in the mutant.
This may be connected to the general overexpression of regulated genes in PCDH12-deficient
placentas. Upregulation of genes of the immune response cluster is more intriguing; it might
reflect a modification in the activity of the uterine natural killer cells of the decidua. More
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generally, it is noteworthy that loss of a protein exhibiting cell-cell adhesive properties has a
significant impact on gene transcription.
Most of the modified genes are expressed by PCDH12-positive cells, i.e., glycogen and
endothelial cells. For example, Gja1 (CX43), Psg19, Mmp9 or Cox1 are solely or primarily
expressed by glycogen cells (5, 6, 23, 36), and TIE1 or KDR are mainly located on
endothelial cell surface (11, 33). This suggests intracellular signaling mechanisms between
the membrane-linked Pcdh12 and the nuclear transcriptional machinery.
Remarkably, the cytoplasmic domains of -protocadherins and protocadherin FAT1 may be
cleaved and translocated to the nucleus (18, 29). This opens interesting prospects for
protocadherin signaling.
Other modified genes are expressed by cell types that do not express PCDH12. For example,
PHLDA2 or the EAR family members are specific of labyrinthine trophoblasts (14) or
immune response cells (24), respectively. This feature indicates that PCDH12 activity may
influence other cell types of the placenta through intercellular signaling mechanisms that
remain to be elucidated.
Angiogenesis
As shown by the histomorphological analysis (Fig. 2), proportions of placenta layers were not
altered in Pcdh12-deficient mice. However, labyrinthine vascular density was decreased (Fig.
4), which may be caused by expression variation of several genes: first, increased levels of the
VEGF decoy receptor gene Flt1 and moreover of its soluble form sFlt1; both are known for
their antiangiogenic properties and are strongly expressed by the junctional zone (11, 13, 20).
Similarly, increased expression of Flt1 and sFlt1 was recently shown to be responsible for the
vascular defect phenotype observed in Adra2b-/- placentas (30); second, upregulation of Tie1,
an orphan receptor regulating endothelial quiescence (34); third, upregulation of Tgfbeta1 and
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2, which are known promoters of vessel maturation; fourth, upregulation of numerous matrix
protease genes, which may release cryptic matrix or non-matrix derived inhibitors of
angiogenesis (see (31), for review); and fifth, upregulation of fibulin 5, which inhibits the
ability of endothelial cells to undergo angiogenic sprouting (2). Some of these anti-angiogenic
factors may also target the maternal vasculature as well (see below).
Placenta morphogenesis and function
Expression of major placental morphogenetic genes, such as Plac1, Pem or Ascl2 was not
significantly modified by loss of PCDH12, with the exception of the imprinted gene Phlda2
(also called Ipl or Tssc3) that was downregulated. Its protein product acts to limit placental
growth in mice. The functional consequences of Phlda2 upregulation is at present unclear.
Genes coding for placental hormones (prolactins) or growth factors (IGF2, EPO) did not show
major alterations in their expression. However, transcription levels of two genes coding for
proteins involved in prostaglandin synthesis and uptake, Ptgs1 (cyclooxygenase 1 or Cox1)
and Slc21a2 (coding for a prostaglandin transporter) respectively, were upregulated.
Prostaglandins are critical molecules for initiation of parturition (reviewed in (32)) and are
currently used to trigger delivery in humans. PCDH12 levels may thus influence the timing of
birth through regulation of prostaglandin activity. This feature may be related to the
upregulation in the mutant placenta of Mmp9 expression, encoding a matrix protease
facilitating tissue remodeling in delivery (25).
We observed altered expression of two members of the pregnancy-specific glycoprotein (Psg)
gene family, Psg16 and Psg19. PSGs are the most abundant fetal proteins in the maternal
bloodstream in late pregnancy (27). In situ hybridization studies showed that Psg19 was
exclusively transcribed by the junctional zone in the mouse (23). The importance of PSGs in
the maintenance of pregnancy has been demonstrated by injection of anti-PSG antibodies,
19
which induced abortion in mice (19). Most studies on PSGs are linked to the modulation of
the maternal immune system, through cytokine secretion induction, preventing rejection of
the fetuses. Consistently, expression of several immune response genes was upregulated in
Pcdh12-/- placentas, including interleukin and chemokine ligands and receptors.
Matrix protein and proteases
Various types of collagen, laminin and fibulin were upregulated in absence of PCDH12. Once
assembled, these secreted proteins are the major constituents of the extracellular matrix where
they have a dual function: they provide tissues with their biomechanical properties and they
support cell guidance during migration. In the placenta, the extracellular matrix enables the
attachment of fetal and maternal tissues and provides a path for trophoblast invasion.
Pericellular protease activity further facilitates cell migration and invasion. Matrix
metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in morphogenesis
of many epitheliomesenchymal organs (review, see (46)). Genes encoding three matrix
metalloproteases, MMP9, 14 and 23, and several members of ADAMs (A Disintegrin And
Metalloproteinase) or ADAMTS (ADAM with ThromboSpondin repeats) families were
upregulated in the PCDH12-deficient placentas. All are involved in cell adhesion, cell
migration, membrane protein shedding and proteolysis. The most upregulated gene of this
category was decysin. This protein is a new member of the ADAM family. In placenta,
decysin is mostly expressed in the junctional zone and around maternal vessels (3), i.e., at
sites of PCDH12 expression. However, its substrate specificity is unknown.
Altogether, the transcriptional upregulation of these gene families is consistent with increased
cell migration and tissue invasion. Therefore, the fact that PCDH12-deficient placentas
showed defects in layer segregation is intriguing. It is possible that the orchestration of the
20
various players of the cell migration process be compromised in the mutant placenta, leading
to uncoordinated cell movement.
RNases
Surprisingly, a number of genes coding for secreted RNases, especially those of EAR 1-3 and
angiogenin 2, were dramatically upregulated in PCDH12-deficient placentas. Some of these
proteins are expressed by neutrophils or macrophages (see (12) and references therein) and
possibly uterine natural killer cells. It has been suggested that these enzymes, as most of the
RNase family members, may be involved in host defense. As indicated for the other immune
response genes whose expression is modified in the mutant placenta, it is possible that
PCDH12 modulates the uterine natural killer cell activity by an unknown mechanism.
Conclusions
Our data show that PCDH12 is a participant in placental morphogenesis. In absence of
PCDH12, placentas exhibited growth retardation and histomorphological alterations. The
gene profiling study comparing wild type and Pcdh12-/- placentas shows expression variations
of a surprisingly large number of genes. This study constitutes a basis for the investigation of
the multiple signaling pathways in which PCDH12 is involved.
21
ACKNOWLEDGMENTS
C. Rampon was supported by the Ligue contre le Cancer and the Fondation pour la Recherche
Médicale. This work was supported by recurrent grants from the Institut National de la Santé
et de la Recherche Médicale, the Commissariat à l’Energie Atomique and Grenoble
University. C. Rampon’s present address : Inserm U770, 94276 Le Kremlin Bicètre, France.
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FIGURE LEGENDS
Figure 1: Placental, embryonic and postnatal weights of PCDH12-deficient mice
Wild type (WT) and Pcdh12-/- placentas and embryos were dissected at E12.5 (A, C, E) (n =
33 and 42, for WT and Pcdh12-/- individuals, respectively) and at E17.5 (B, D, F) (n = 46 and
33 for WT and Pcdh12-/- individuals, respectively). Placentas were weighed and their volume
were calculated after measurement with a caliper, as described in Materials and Methods.
Embryos were weighed after elimination of placenta and yolk sac. Histograms show (A, B)
placenta weights, (C, D) placenta volumes, and (E, F) embryonic weights. Litter-mates from
heterozygous X heterozygous matings were weighed from 2 to 8 weeks of postnatal age.
Histograms in (G, H) represent postnatal weights of males (G) (n = 5 and 7, for WT and
PCDH12-deficient mice, respectively) and females (H) (n = 7 and 6, for WT and PCDH12deficient mice, respectively). Data represent the mean with SD. p-values are indicated when
statistically significant differences were present.
Figure 2: Histomorphological analysis of Pcdh12-/- placentas and deciduas
(A) Parasagittal sections of placentas (shown here at E17.5) were stained with anti-CD31
antibody (brown) and hematoxylin (blue) to visualize the anatomical layers: the labyrinth (L),
the junctional zone (J) and the decidua (D). Lines indicate layer separations. At this
magnification, individual vessels are not visible, but vascular zones appear brownish. (B, C)
The surfaces of the 3 layers, as well as total surfaces, were measured on 3 parasagittal
sections (separated by approximately 50 µm) for each placenta at E12.5 (B) and E17.5 (C).
The mean values were used to calculate the mean surface proportion (SD) of layers from
wild-type (n = 8 and 12, for E12.5 and E17.5 placentas, respectively) and Pcdh12-/- (n = 8 and
10 for E12.5 and E17.5 placentas, respectively) placentas. (D, E) For each placenta at E12.5
(D) and E17.5 (E), nuclei were counted on three different areas at high magnification to
29
calculate the cell density. Data represent the mean with SD. A significant difference was
observed for labyrinthine cell density in both E12.5 and E17.5 placentas.
Figure 3: Measurement of total capillary length in placenta histological sections
Histological sections of wild type and Pcdh12-/- E12.5 (n = 5 for each) and E17.5 (n = 6 for
each) placentas were labeled with anti-CD31 antibody and visualized by immunofluorescence
microscopy at high magnification. Labyrinthine vascular density was decreased in Pcdh12-/placentas compared to wild types, as shown for E17.5 placentas (A, B). For quantifications,
three different images of labyrinth were acquired for each placenta. Images were binarized
and skeletonized and total capillary length was measured as described in Material and
Methods. (C) Initial CD 31 fluorescent image. (D) Processed image. (E) Merge. (F, G) The
data show the average length (SD) of the capillary network per labyrinth area for each
genotype at E112.5 (F) and E17.5 (G). CD31-labeled deciduas for which mother and embryo
were of same genotype, i.e., wild type or Pcdh12-/-. Vascular patterns of wild type (H) and
Pcdh12-/- (I) E12.5 deciduas were similar.
Figure 4: Glycogen cell islets in the junctional zone and the labyrinth of Pcdh12-/- placentas
PAS staining and hematoxylin counterstaining of wild type (A,C) and Pcdh12-/- (B,D,E)
placenta sections. Glycogen cells are stained in deep purple by PAS because of their high
glycogen contents. (A,B) E12.5 placenta section images are focused on the junctional zone.
Glycogen cell islets are circled and indicated with (*). Note that glycogen cells appear
partially empty because of glycogen solubilization occuring during section preparation. (C,D)
E17.5 total placenta sections are shown. At this age, the labyrinth constitutes the major part of
the placenta; the junctional zone and the decidua form a cap at the placenta margin. (E) is an
enlargement of (D) showing that junctional zone islets (circled) are composed of both cell
29
30
types. Sp, spongiotrophoblast; GC, glycogen cells. (F) The number of islets (pegs) was
averaged in sections from wild type (n = 9) and Pcdh12-/- (n = 12) placentas. The error bars
represent the SE.
Figure 5: Glycogen content of Pcdh12-/- placentas
E12.5 (A, C) and E17.5 (B, D) placentas were weighed and glycogen was extracted as
described in Materials and Methods. Glycogen content was measured by a colorimetric assay
using mussel glycogen as standard. Glycogen concentration (A, B) and glycogen amount (C,
D) at E12.5 (n = 41 and 32 for wild type and Pcdh12-/- placentas, respectively) and at E17.5 (n
= 31 and 33 for wild type and Pcdh12-/- placentas, respectively) are shown. Data represent the
mean with SE.
30
31
Table 1: Confirmation by qRT-PCR of representative gene expression in Pcdh12-/- placenta
compared to wild type
qRT-PCR†
WT/Pcdh12-/-
Microarray
1,230
ratio
0.56
ratio
0.50
360,815
355,650
0.99
1.03
1,021
1,017
1.00
1.08
Tek (Tie 2)
122
171
1.40
1.58
Tie1
177
364
2.06
2.55
Gja1 (Cx43)
22,436
79,117
3.53
4.29
Angiogenin 2
103
4,808
47
102
Decysin
19
9,137
491
180
Gene Symbol*
WT
Pcdh12-/-
Phdal2 (Ipl)
2,188
Gapdh
Angiopoietin 2
*Genes were chosen for their different expression levels and fold variations according to
DNA chips data. †Data are expressed in arbitrary units and represent the mean value for 5
placentas in each case, normalized by Gapdh values, except for Gapdh.
31
32
Table 2: Changes in gene expression categorized by gene ontology
Changes
Total Under Over FWER value†
Gene Cluster*
Development and tissue morphogenesis
485
0
144
0.010
Cell adhesion and migration, matrix proteins
80
0
62
0.010
Immune response
216
0
39
0.000
Angiogenesis
47
0
16
0.040
Chromatin
70
9
0
0.040
1
Gene grouping was obtained through GoMiner software and genes were further gathered
in 5 clusters. †The detection of over and under expressed genes for each cluster and the
corresponding Family Wise Error Rate was obtained with Gene Set Enrichment Analysis
procedure (see Materials and Methods).
32
33
Table 3: Changes in expression of specific placental, junctional or angiogenic genes*
Gene
Symbol
Gene Product†
WT
Pcdh12-/- Pcdh12-/Signal
Signal WT Signal SAM
Affymetrix ID Average‡ Average‡
Ratio
Score Q-Value
Placental genes
Phlda2 Pleckstrin homology-like domain, family A, member 2
(IPL, TSSC3)
Tpbpa Trophoblast specific protein alpha
Ascl2
Achaete-scute complex homolog-like 2 (MASH2)
Plac1 Placental specific protein 1
Pem
Placentae and embryos oncofetal gene
Prlpb
Prolactin-like protein B
Prlpc
Prolactin-like protein C (PLP-H)
Prlpcb Prolactin-like protein C beta
Prlpcg Prolactin-like protein C-gamma (PLP-D)
Upar2 Urokinase-type plasminogen activator receptor,type 2
Epo
Erythropoietin 1
Igf2
Insulin-like growth factor 2
Igf2r
Insulin-like growth factor 2 receptor
Ptgs1
Prostaglandin-endoperoxide synthase 1 (COX1)
Ptgs2
Prostaglandin-endoperoxide synthase 2 (COX2)
Slc21a2 Solute carrier family 21 (prostaglandin transporter)
1417837_at 2252.22
1415808_at 17589.14
1422396_s_at
178.82
1417553_at 2345.84
1423429_at 4379.28
1420571_at 4874.54
1418418_a_at
588.28
1448532_at 13424.94
1424387_at 2249.24
1452521_a_at
176.28
1433508_at
545.18
1415931_at
71.14
1424112_at
254.76
1423414_at
21.06
1417262_at
60.24
1420913_at
507.48
Psg16
Psg19
1449238_at
1421418_a_at
Protocadherin 12
1450473_at
E-cadherin
1448261_at
N-cadherin
1418815_at
P-cadherin
1426673_at
VE-cadherin
1433956_at
Gap junction membrane channel protein alpha 1 (CX43) 1438650_x_at
Gap junction membrane channel protein beta 2 (CX26)
1423271_at
Gap junction membrane channel protein beta 3 (CX31)
1416715_at
Claudin 1
1437932_a_at
Claudin 5
1417839_at
Claudin 11
1416003_at
Pregnancy specific GP16
Pregnancy specific glycoprotein 19
1132.68
15863.9
134.86
1780.32
3564.46
2649.18
729.88
12016.8
2006.56
205.1
576.22
112.06
387.14
64.88
66.24
1280.96
0.50
0.90
0.75
0.76
0.81
0.54
1.24
0.90
0.89
1.16
1.06
1.58
1.52
3.08
1.10
2.52
2.75
0.36
0.35
0.48
0.61
0.84
0.39
0.48
0.16
0.33
0.16
0.58
0.68
0.95
0.16
1.74
378.72
39.78
153.34
175.06
0.40
4.40
0.85
1.28
0.059
0.285
0.212
0.156
0.105
0.143
0.317
0.309
0.532
0.173
0.524
0.100
0.175
0.108
0.506
0.046
0.095
0.068
64.96
744.42
14.24
499.22
496.2
706.08
3194.64
349.7
112.42
170.6
39.26
6.92
680.3
28.36
775.92
1024.34
3031.44
3498.7
290.74
296.88
235.88
107.26
0.11
0.91
1.99
1.55
2.06
4.29
1.10
0.83
2.64
1.38
2.73
1.32
0.11
0.69
0.80
1.38
2.17
0.17
0.57
0.97
1.25
0.88
0.089
0.524
0.182
0.158
0.083
0.063
0.511
0.149
0.079
0.103
0.099
Cell junction genes
Pcdh12
Cdh1
Cdh2
Cdh3
Cdh5
Gja1
Gjb2
Gjb3
Cldn1
Cldn5
Cldn11
Angiogenic genes
Vegfa Vascular endothelial growth factor A
1420909_at
97.88
102.56
1.05
0.06
0.581
Pgf
Placental growth factor
1418471_at
163.88
202.56
1.24
0.42
0.318
Fgfr2
Fibroblast growth factor receptor 2
1433489_s_at
332.46
244.36
0.74
0.73
0.185
Fgfr1
Fibroblast growth factor receptor 1
1424050_s_at
327.44
637.68
1.95
1.16
0.068
Tie1
Tyrosine kinase receptor 1
1416238_at
35.26
89.76
2.55
0.90
0.143
Tek
Endothelial-specific receptor tyrosine kinase
1418788_at
69.82
110.1
1.58
1.03
0.087
Eng
Endoglin
1417271_a_at
108.98
201.18
1.85
1.69
0.046
Angpt2 Angiopoietin 2
1448831_at
789.16
855.16
1.08
0.08
0.558
Alk1
Activin A receptor, type II-like 1
1451604_a_at
103.24
160.12
1.55
0.82
0.143
Tgfb1 Transforming growth factor, beta 1
1420653_at
86.22
191.68
2.22
0.95
0.102
Tgfb2 Transforming growth factor, beta 2
1438303_at
34.78
95.78
2.75
1.51
0.057
Tgfbr1 Transforming growth factor, beta receptor I
1420895_at
255.16
275.84
1.08
0.25
0.417
Hif1a
Hypoxia-inducible factor one alpha
1427418_a_at
1340.2
1431.92
1.07
0.22
0.399
sFlt1
Soluble FMS-like tyrosine kinase 1
1451756_at
976.78
1673.66
1.71
0.71
0.143
Flt1
FMS-like tyrosine kinase 1
1419300_at
197.68
294.3
1.49
0.51
0.143
Kdr
Kinase insert domain protein receptor
1449379_at
75.66
130.66
1.73
0.87
0.103
*Genes of these categories were selected as follows: lowest average signal value above 15 (except Pcdh12). †Other designations are
indicated in parenthesis. ‡In arbitrary units.
33
34
Table 4: Changes in expression of genes involved in cell-matrix adhesion and migration*
Gene
Symbol
Gene Product
Matrix and matrix-related genes
Col1a1
Procollagen, type I, alpha-1
Col1a2
Procollagen, type I, alpha 2
Col4a2
Procollagen, type IV, alpha 2
Col5a1
Procollagen, type V, alpha 1
Col5a2
Procollagen, type V, alpha 2
Col6a3
Procollagen type VI, alpha 3
Col15a1 Procollagen, type XV, alpha 1
Procollagen lysine, 2-oxoglutarate 5Plod2
dioxygenase 2
Lox
Lysyl oxidase
Lama1
Laminin, alpha 1
Lama5
Laminin, alpha 5
Lamb2
Laminin, beta 2
Fbn1
Fibrillin 1
Fbln2
Fibulin 2
Fbln5
Fibulin 5
Integrin genes
Itga3
Integrin alpha 3
Itga6
Integrin alpha 6
Itgav
Integrin alpha V
Itgb1
Integrin beta1D
Itgb5
Integrin beta 5
Itgb7
Integrin beta 7
Affymetrix ID
WT
Signal
Average
Pcdh12-/- Pcdh12-/Signal WT Signal
Average
Ratio
SAM
Score
Q-Value
1423669_at
1450857_a_at
1424051_at
119.52
359.84
904.66
364.06
996.74
2044.74
3.05
2.77
2.26
1.15
1.53
1.16
1416740_at
1450625_at
1424131_at
1426955_at
90.32
153.46
38.94
67.24
306.74
551.64
131.54
150.2
3.40
3.59
3.38
2.23
1.75
1.27
1.27
1.35
0.115
0.087
0.096
0.078
0.068
0.095
0.058
1416687_at
1416121_at
1418153_at
1427009_at
1416513_at
1425896_a_at
1423407_a_at
1416164_at
316.70
337.24
113.22
312.54
126.9
66.12
131.9
50.04
990.40
893.10
349.60
706.98
290.38
182.16
324.54
181.10
3.13
2.65
3.09
2.26
2.29
2.75
2.46
3.62
1.15
0.78
0.77
1.83
1.45
0.98
1.04
1.26
0.066
0.112
0.165
0.078
0.090
0.089
0.083
0.061
1421997_s_at
1422444_at
1421198_at
1452545_a_at
1417533_a_at
1418741_at
49
205.18
165.18
696.92
755.58
22.88
140.4
292.2
221.82
916.86
1392.96
46.88
2.87
1.42
1.34
1.32
1.84
2.05
1.45
1.17
0.74
1.01
1.17
0.76
0.082
0.124
0.093
0.098
0.075
0.152
1.78
0.88
0.93
0.61
0.068
0.141
0.087
0.238
0.83
0.068
0.50
0.189
0.92
0.098
0.90
0.093
0.85
0.118
0.77
0.81
0.143
0.103
Matrix protease and matrix protease inhibitor genes
Dcsn
Disintegrin metalloprotease (decysin)
1419476_at
8.90
1598.20
179.57
Mmp9
Gelatinase
1416298_at
15.0
35.44
2.41
Mmp14 Matrix metalloproteinase 14
1448383_at
189.3
396.1
2.09
Mmp23 Matrix metalloproteinase 23
1417281_a_at
14.44
43.98
3.05
A disintegrin and metalloproteinase
Adam9
domain 9
1416094_at
233.4
335.9
1.44
A disintegrin and metalloproteinase
Adam12 domain 12
1421171_at
58.06
100.96
1.74
A disintegrin and metalloproteinase
Adam17 domain 17
1421858_at
208.26
262
1.26
A disintegrin and metalloprotease
Adam19 domain 19
1418403_at
196.76
385.88
1.96
Adamts2 A disintegrin-like and metalloprotease
with thrombospondin type 1 motif, 2
1457058_at
248.08
562.9
2.27
Adamts9 A disintegrin-like and metalloprotease
with thrombospondin type 1 motif, 9
1431399_at
28.76
47.94
1.67
Timp2
Tissue inhibitor of metalloproteinase 2
1450040_at
39.44
74.6
1.89
*Genes of these categories were selected as follows: lowest signal value above 15, SAM score above 0.50.
34
35
Table 5: Changes in expression of miscellaneous genes*
Gene
Symbol
Gene Product
RNA-related genes
Ear1
Eosinophil-associated ribonuclease 1
Ear2
Eosinophil-associated ribonuclease 2
Ear3
Eosinophil-associated ribonuclease 3
RNasea4 RNase A family 4
Ang2
Angiogenin 2, ribonuclease A family
Rbm3
RNA-binding motif protein 3
Affymetrix ID
WT
Signal
Average
Pcdh12-/- Pcdh12-/Signal WT Signal
Average
Ratio
SAM
Score
Q-Value
1421802_at
1449846_at
1422411_s_at
1422603_at
1422415_at
1422660_at
2.90
24.56
161.88
527.26
2.72
3490.62
690.48
2367.70
1810.56
1475.30
278.22
1505.94
238.10
96.40
11.18
2.80
102.29
0.43
3.78
2.74
2.03
0.92
1.51
1.13
0.046
0.055
0.065
0.099
0.075
0.100
1416625_at
1418762_at
1417266_at
1419209_at
1418536_at
997.34
130.74
47.24
33.62
45.9
2616.52
374.96
107.36
100.06
111.62
2.62
2.87
2.27
2.98
2.43
0.75
0.87
1.03
2.15
1.04
0.121
0.068
0.110
0.068
0.068
1420394_s_at
1423547_at
1450199_a_at
50.46
91.7
55.82
105.52
188.56
127.52
2.09
2.06
2.28
0.84
0.91
0.92
0.153
0.150
0.106
Immune response genes
C1i
Daf1
Ccl6
Cxcl1
H2-Q7
Lilrb4
Lyzs
Stab1
Complement component 1 inhibitor
Decay accelerating factor 1
Chemokine (C-C motif) ligand 6
Chemokine (C-X-C motif) ligand 1
Histocompatibility 2, Q region locus 7
Leukocyte immunoglobulin-like
receptor, subfamily B, member 4
Lysozyme
Stabilin
Unrelated genes
Hmgb2
High mobility group box 2
1437313_x_at
843.62
356.00
0.42
1.07
0.098
Lbp
Lipopolysaccharide binding protein
1448550_at
516.62
1815.60
3.51
0.90
0.099
Hk2
Hexokinase 2
1422612_at
260.28
721.66
2.77
1.19
0.082
Gsn
Gelsolin
1456312_x_at 607.50
2428.50
4.00
1.23
0.091
Cyclin E2 Cyclin E2
1422535_at
415.10
170.10
0.41
0.97
0.123
*Genes were selected as follows: Pcdh12-/- / WT signal ratio above 2 or below 0.5, highest average value above 100, SAM
score above 0.8. Genes shown in Tables 3-4 were excluded.
35